Methods for single molecule sequencing

Abstract

Provided herein are methods and systems for sequencing a single nucleic acid molecule utilizing a polymerase enzyme, a template nucleic acid, and a polymerase reagent solution.

What is claimed is: 
     
       1. A method for sequencing a nucleic acid template comprising:
 providing a sequencing mixture comprising (i) a polymerase enzyme, (ii) a template nucleic acid, and (iii) a polymerase reagent solution having the components for carrying out template directed synthesis of a growing nucleic acid strand, wherein said polymerase reagent solution includes a component for a requenching reaction and a plurality of types of nucleotide analogs; wherein each type of nucleotide analog has a labeled leaving group that is cleavable by the polymerase, and each type of nucleotide analog has a different label, wherein the labeled leaving group is cleaved upon polymerase-dependent binding of a respective nucleotide analog to the template strand; 
 carrying out nucleic acid synthesis such that a plurality of nucleotide analogs are added sequentially to the template whereby: a) a nucleotide analog associates with the polymerase, b) the nucleotide analog is incorporated on the template strand by the polymerase when the labeled leaving group on that nucleotide analog is cleaved by the polymerase, wherein the labeled leaving group emits light upon cleavage, then c) the labeled leaving group on the nucleotide analog is quenched by the requenching reaction, wherein a removable quencher molecule is attached to the nucleotide analog through electrostatic interaction or hydrogen bonding to achieve dual-quenching; and 
 detecting light from the labels while nucleic acid synthesis is occurring, and using light detected in the time between step b) when the labelled leaving group is cleaved, and step c) in which the labeled leaving group is quenched, to determine a sequence of the template nucleic acid. 
 
     
     
       2. The method of  claim 1 , wherein the nucleotide analog has been modified by a fluorophore attached to a terminal phosphate. 
     
     
       3. The method of  claim 1 , wherein the leaving group is a labelled pyrophosphate. 
     
     
       4. The method of  claim 3 , wherein the pyrophosphate is labeled with a fluorophore. 
     
     
       5. The method of  claim 1 , wherein each base of a nucleotide is labeled with a unique fluorophore relative to other bases. 
     
     
       6. The method of  claim 1 , wherein the requenching reaction uses a quenching enzyme or Plasmonics. 
     
     
       7. The method of  claim 6 , wherein the quenching enzyme is ATP sulfurylase. 
     
     
       8. The method of  claim 1 , wherein requenching is achieved by an adenosine nucleobase on APS. 
     
     
       9. The method of  claim 1 , wherein the polymerase enzyme is DNA polymerase. 
     
     
       10. The method of  claim 1 , wherein types of nucleotide analogs comprise dATP, dTTP, dGTP, dCTP and dUTP. 
     
     
       11. The method of  claim 1 , wherein the requenching reaction uses plasmonics. 
     
     
       12. The method of  claim 2 , wherein the fluorophore is quenched by the nucleobase on the nucleotide. 
     
     
       13. The method of  claim 1 , wherein the removable quencher molecule is attached to the nucleotide analog at the nucleobase or sugar.